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1.
Replenishment of medium after 72 hr of growth of HeLa-S3 cells in dense suspension cultures increased [3H]-thymidine uptake into cells and incorporation into DNA, with the levels reaching a peak ~ 12 hr following medium change; β interferon inhibits the enhanced uptake of [3H]-thymidine and labeling of DNA in a dose-dependent manner. Some reduction in these processes is observed at a concentration as low as 1 u/ml, and ~ 75% inhibition at 640 u/ml. Kinetic analysis has revealed that the rate of labeling of the acid-soluble pool with [3H]-thymidine, measured either at 22°C, or 37°C, is reduced in interferon-treated (640 u/ml, 24 hr) HeLa-S3 cells. At 22°C, the initial rate of thymidine transport at a high (500 μM) thymidine concentration, determined within the first 30 sec of [3H]-thymidine addition was depressed by 44% in interferon-treated HeLa cells. At 37°C, labeled precursors accumulate in acid-soluble material for ~ 8 min after the addition of [3H]-thymidine, after which an apparent equilibrium level is attained. At this temperature, the rate of thymidine uptake and the apparent equilibrium level attained were depressed by 70% in interferon-treated HeLa cells. The reduced incorporation of [3H]-thymidine into DNA in interferon-treated HeLa-S3 cells can be largely explained by interferon inhibition of thymidine transport and phosphorylation. 相似文献
2.
B.E. Linebaugh J.A. Rillema 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,720(4):346-355
Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [3H]uridine incorporation into RNA and [3H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10−21 M). Insulin stimulated the rate of [3H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [3H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [3H]uridine, [3H]thymidine and [3H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis. 相似文献
3.
To determine the respective role of thymidine kinase and thymidylate synthase activities in the hyperoxia-induced decrease in DNA synthesis and their relationship with cell replication, we measured these two enzyme activities in primary cultures of porcine aortic endothelial cells under different O2 concentrations for various durations. In confluent cells, exposure to 95% O2 for 5 days reduced thymidine kinase activity to 15% of control values; thymidylate synthase activity was unaffected. In preconfluent cells exposed to 95% O2 for 2 days, similar results were obtained, together with evidence for arrest in cell proliferation. Thymidylate synthase activity could therefore not be related to decreased cell proliferation under hyperoxia. [3H]thymidine incorporation into DNA, thymidine kinase activity, and cell proliferation were all similarly affected under exposure to graded O2 concentration for 2 days. Thymidine kinase appears to be a key enzyme in the modulation of DNA synthesis from thymidine and in its replication in endothelial cells. 相似文献
4.
Summary Cultured cells from the bovine endosalpinx were used to evaluate effects of estradiol-17β, progesterone, epidermal growth
factor, and insulinlike growth factors I and II on [3H]thymidine incorporation. Cells were treated with hormones and growth factors when approximately 50% confluent. After 24
h, DNA synthesis was quantified by pulsing cells with [3H]thymidine for 12 h and determining uptake into DNA. Cells prepared by mechanical dispersal incorporated more [3H]thymidine than cells dispersed with collagenase. However, hormonal responses were the same for both types of cells. As compared
to plastic, cells on a Matrigel substratum exhibited lower incorporation of [3H]thymidine and were unresponsive to hormones. Estradiol-17β increased [3H]thymidine incorporation slightly at 10−10 mol/liter and higher. Epidermal growth factor, insulinlike growth factor-I, and insulinlike growth factor-II also stimulated
[3H]thymidine incorporation. Effects of insulinlike growth factor-I were greater for cells treated with estradiol-17β. In the
absence of estradiol, progesterone inhibited [3H]thymidine incorporation at 1, 10, and 100 ng/ml. When estradiol-17β was present, progesterone stimulated [3H]thymidine incorporation at 1 ng/ml and reduced incorporation at 100 ng/ml. In conclusion, [3H]thymidine incorporation by cultured oviductal endosalpingeal cells can be regulated by ovarian steroids and growth factors.
These molecules may represent signals through which the ovary, embryo, and oviduct regulate oviductal growth.
Work conducted while on a sabbatical leave supported by the Deutsche Forschungsgemeinschaft. 相似文献
5.
DNA replication in Escherichia coli: evidence for two classes of small deoxyribonucleotide chains 总被引:8,自引:0,他引:8
We have used [3H]thymidine to pulse label cultures growing at 20 or 37 °C. At these temperatures, thymidine can be used interchangeably with thymine for labeling periods of 0.1 minute or longer. Incorporation does not stop immediately when cultures are poured on to ice-cold medium-KCN mixtures, but can be stopped by pouring on to the same mixture plus pyridine.We have extracted DNA from cells pulse-labeled with [3H]thymidine and measured the number of short deoxynucleotide chains by treating them with bacterial alkaline phosphatase and labeling their 5′ ends with 32P using [γ-32P]ATP and polynucleotide kinase. There are at least 40 deoxynucleotide chains per bacterium (20 per replicating chromosome) whose sedimentation coefficient is less than 15 S.The small deoxynucleotide chains labeled by a pulse of [3H]thymidine behave as intermediates in the replication of DNA. They accumulate if ligase is inhibited and they disappear if their synthesis is blocked by inactivating a gene product responsible for their synthesis. In contrast, the 32P-labeled pieces do not accumulate rapidly or disappear under the same experimental conditions. The data indicate that many of the 32P-labeled short chains are not located at the replication fork and that they do not behave as intermediates in the replication of DNA.The size distribution of3H pulse-labeled pieces and of the short chains labeled with 32P are apparently the same when measured by sedimentation through alkaline sucrose. About 25% of the molecules are extremely short. The remainder are distributed in such a way that, roughly, the number of pieces greater than any particular length decreases exponentially as that length increases. 相似文献
6.
Selected Nucleic Acid Precursors in Studies of Aquatic Microbial Ecology 总被引:17,自引:13,他引:4
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David M. Karl 《Applied microbiology》1982,44(4):891-902
The use of radiolabeled nucleosides and nucleic acid bases to estimate the rates of RNA and DNA synthesis in naturally occurring microbial assemblages requires numerous assumptions, several of which are evaluated herein. Comparative time series analyses of the uptake and incorporation, labeling specificity, and extent of catabolism of [2-3H]adenine, [methyl-3H]thymidine, and [5-3H]uridine were performed with pure bacterial and algal cultures, as well as with environmental samples. [3H]thymidine yielded the most variable results, especially with regard to the extent of nonspecific macromolecular labeling. The pathways of [3H]thymidine and [3H]adenine metabolism were further evaluated by isotope dilution methods and by comparing incorporation patterns of thymidine labeled at different sites of the molecule. The advantages, uncertainties, and limitations of the use of radiolabeled nucleic acid precursors in studies of aquatic microbial ecology are discussed and a prospectus for future studies presented. 相似文献
7.
Nino Sorgente Debra L. Bullard Ljiljana Jakovljevic Kathleen Dorey 《Cell proliferation》1984,17(6):573-582
Bovine aortic endothelial cells (BAEC) in culture have the ability to regulate their own proliferation. We have found that a fraction below 100,000 daltons obtained from the media of confluent cultures of BAEC inhibits tritiated thymidine [3H]TdR incorporation as well as their proliferation. the inhibition is dose- and time-dependent; maximum inhibition of [3H]TdR incorporation occurs 8 hr after cells are released from synchronization and the inhibitory fraction is added. Inhibition is evident at concentrations as low as 50 μg/ml and reaches a maximum at 600 μg/ml. the blockage of [3H]TdR incorporation is reflected in the inhibition of cell proliferation. In the presence of 400 μg of endogenous inhibitor per ml of media, added at the time of plating, the average population doubling time increases from 19 to 41 hr. These findings indicate that, in culture, BAEC can regulate their own proliferation by synthesizing an endogenous inhibitor(s) of proliferation. 相似文献
8.
The purpose of this experiment was to determine the effect of ionizing radiation on cell number, lactate dehydrogenase (LDH) release, cell cycle distribution, [3H]thymidine incorporation, and autoradiographic labeling index in bovine aortic endothelial cells in vitro. Confluent endothelial monolayers were exposed to single doses of 0.5-10 Gy of 60Co gamma rays and were analyzed from 2 to 24 h postirradiation. Irradiated monolayers exhibited a time- and dose-dependent decrease in cell number, increase in LDH release, and redistribution of cells in the cell cycle. Cell cycle redistribution included an increase in the proportion of cells in S phase at 4 h after irradiation and a decrease in S phase at 24 h. The cells also exhibited a decrease in [3H]thymidine incorporation as early as 2 h after 5 Gy. This represented the most rapid radiation response observed in the present study. These data demonstrate that radiation cytotoxicity in confluent, plateau-phase endothelial monolayers is accompanied by changes in the cell cycle distribution of adherent cells, and that reduced [3H]thymidine incorporation is an early marker of radiation injury in this clinically important cell type. 相似文献
9.
A direct comparison of [3H]thymidine incorporation with DNA synthesis was made by using an exponentially growing estuarine bacterial isolate and the naturally occurring bacterial populations in a eutrophic subtropical estuary and in oligotrophic offshore waters. Simultaneous measurements of [3H]thymidine incorporation into DNA, fluorometrically determined DNA content, and direct counts were made over time. DNA synthesis estimated from thymidine incorporation values was compared with fluorometrically determined changes in DNA content. Even after isotope dilution, nonspecific macromolecular labeling, and efficiency of DNA recovery were accounted for, [3H]thymidine incorporation consistently underestimated DNA synthesized by six- to eightfold. These results indicate that although the relationship of [3H]thymidine incorporation to DNA synthesis appears consistent, there are significant sources of thymine bases incorporated into DNA which cannot be accounted for by standard [3H]thymidine incorporation and isotope dilution assays. 相似文献
10.
Using pulse labeling techniques with [3H]thymidine or [3H]cytidine, combined with DNA fiber autoradiography, we have investigated the direction and rate of DNA chain growth in mammalian cells. In general, chain elongation proceeds bidirectionally from the common origin of pairs of adjacent replication sections. This type of replication is noted whether the DNA is labeled first with [3H]thymidine of high specific activity, followed by [3H]thymidine of low specific activity or the sequence is reversed. Approximately one-fifth of the growing points have unique origins and in these replication units, chain growth proceeds in one direction only. Fluorodeoxyuridine and hydroxyurea both inhibit DNA chain propagation. Fluorodeoxyuridine exerts its effect on chain growth within 15–23 min, while the effect of hydroxyurea is evident within 15 min under conditions where the endogenous thymidine pool has been depleted by prior treatment with fluorodeoxyuridine. Puromycin has no effect on chain growth until 60 min after addition of the compound, even though thymidine incorporation is more than 50% reduced within 15 min. After 2 h of treatment with puromycin, the rate of chain growth is reduced by 50%, whereas thymidine incorporation is reduced by 75%. Cycloheximide reduces the rates of DNA chain growth and thymidine incorporation 50% within 15 min, and, on prolonged treatment, the decrease in rate of chain growth generally parallels the reduction in thymidine incorporation. 相似文献
11.
Evidence for an endothelial cell-derived factor which stimulates the growth of human peripheral blood mononuclear cells 总被引:1,自引:0,他引:1
Evidence is provided which demonstrates that conditioned media of cultured endothelial cells derived from human umbilical veins contained a factor which stimulated peripheral blood mononuclear cell [3H]thymidine uptake. A dose-dependent response in peripheral blood mononuclear cell [3H]thymidine uptake was obtained when cells were incubated with increasing concentrations of supernatant of endothelial cell cultures. Studies on temporal kinetics demonstrated that stimulatory activity was evident when mononuclear cells had been incubated with endothelial cell supernatant for 120 hr or more. Preliminary characterization showed the growth immunoregulatory factor to have a molecular weight greater than 100,000 Da. 相似文献
12.
I. M. Drobysheva 《Hydrobiologia》1986,132(1):189-193
Autoradiography has been applied to two acoel turbellarians, Convoluta convoluta and Oxyposthia praedator, to determine the distribution and fate of proliferative cells. In C. convoluta, mitotic figures and nuclei that labelled with [3H]thymidine could be observed in the peripheral parenchyma but not in the middle zone of the central parenchyma. The time required for regeneration of physiologically competent digestive cells was about 10–15 days. In O. praedator, mitotic figures (in metaphase and telophase) were observed in the peripheral parenchyma while none were found in the epidermis either in untreated animals or after treatment with colchicine. Mitotic figures were found only rarely in the central parenchyma and only in its marginal zone. Autoradiographs of O. praedator demonstrated [3H]thymidine incorporation into both the nuclei and the cytoplasm of peripheral parenchymal cells. In the central parenchyma, no nuclei with primary labelling were observed. The digestive parenchyma of the acoels is regarded as a unique histological system involving both specialized cells of the central parenchyma and stem cells located in the peripheral parenchyma. 相似文献
13.
Effect of 5-Fluoro-2′-Deoxyuridine on [3H]Thymidine Incorporation by Bacterioplankton in the Waters of Southwest Florida
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The effect of 5-fluoro-2′-deoxyuridine (FdUrd) on [methyl-3H] thymidine incorporation by bacterioplankton populations in subtropical freshwater, estuarine, and oceanic environments was examined. In estuarine waters, intracellular isotope dilution was inhibited by FdUrd, which enabled us to estimate both intracellular and extracellular isotope dilution. In 2 of 10 cases, extracellular isotope dilution was significant. At low concentrations of [methyl-3H]thymidine or [6-3H]thymidine, FdUrd completely inhibited incorporation of radioactivity into protein and RNA. At high concentrations of [3H]thymidine, however, FdUrd had little effect on labeling patterns. The dihydrofolate reductase inhibitors amethopterin and trimethoprim had no effect on macromolecular labeling patterns. These results suggest that thymidylate synthase is not involved in nonspecific labeling and that FdUrd inhibits nonspecific labeling by blocking some other enzyme involved in thymidine catabolism. In oligotrophic oceanic and freshwater samples, FdUrd did not inhibit intracellular isotope dilution or [3H]thymidine labeling of protein and RNA, but caused some inhibition of [3H]thymidine incorporation into DNA. The ability of FdUrd to inhibit nonspecific macromolecular labeling during [3H]thymidine incorporation was significantly correlated (r = 0.84) with total thymidine incorporation (in picomoles per liter per hour). The results are discussed in terms of applications of FdUrd to routine bacterial production measurements and the general assumptions of [3H]thymidine incorporation. 相似文献
14.
《Mutation Research/DNA Repair Reports》1987,183(3):249-256
We have investigated the effects of fluctuations in deoxynucleoside triphosphate (dNTP) pool size on DNA repair and, conversely, the effect of DNA repair on dNTP pool size. In confluent normal human skin fibroblasts, dNTP pool size was quantitated by the formation of [3H]TTP from [3H]thymidine; DNA repair was examined by repair replication in cultures irradiated with UV light. As defined by HPLC analysis, the [3H]TTP pool was formed within 30 min of the addition of [3H]thymidine and remained relatively constant for the next 6 h. Addition of 2–10 mM hydroxyurea (HU) caused a gradual 2–4-fold increase in the [3H]TTP pool as HU inhibited DNA synthesis but not TTP production. No difference was seen between the [3H]TTP pool size in cells exposed to 20 M/m2 and unrradiated controls, although DNA-repair synthesis was readily quantitated in the former. This result was observed even though the repair replication protocol caused an 8–10-fold reduction in the size of the [3H]TTP pool relative to the initial studies. In the UV excision-repair studies the precense of hydroxyurea did not alter the specific activity of [3H] thymidine 5'-monophospahte incorporated into parental DNA due to repaier replication. These results suggest that fluctuations in the deoxynucleoside triphosphate pools do not limit the extent of excision-repair sythesis in human cells and demonstrate that DNA nucleotide excision-repair synthesis does not significantly diminish the size of the [3H]TTP pool. 相似文献
15.
Thomas C. Mayer 《Developmental biology》1980,79(2):419-427
Cultures of 14-day embryonic mouse epidermis that include melanoblasts initiate melanin synthesis 30 hr after plating, a schedule that is 2.5 days earlier than in vivo. In order to determine if the accelerated differentiation of melanoblasts is related to a cessation of cell proliferation in the cultures, a study of [3H]thymidine incorporation by melanoblasts and melanocytes was made. Autoradiograms of 14-day epidermal cultures grown for 48 hr in medium containing [3H]thymidine revealed that melanoblasts continue to proliferate during this time period. A second population of melanoblasts that did not incorporate [3H]thymidine was also present in these cultures. The relative numbers of dividing and nondividing melanoblasts change with the age of the epidermis cultured. Ninety-one percent of the melanoblasts in 13-day epidermis take up [3H]thymidine, 63% incorporate [3H]thymidine in 14-day cultures, and only 29% take up label in cultures of 15-day epidermis. It appears from these results that melanoblasts during their migration from the neural crest are proliferative cells and that during the early invasion of the epidermis a nonproliferative population of melanoblasts is established. Both populations coexist in the epidermis and subsequently undergo differentiation on the same time schedule. 相似文献
16.
Porcine skin nucleoplasmic extract (PSNE) was shown to alter the incorporation of [3H]thymidine into DNA of selected porcine, bovine, and human cell populations in culture. PSNE stimulated incorporation of
[3H]thymidine into DNA of porcine and bovine dermal cells an average of 300 and 200% of control value, respectively. When porcine
and bovine epidermal cells were exposed to PSNE the treatment inhibited [3H]thymidine incorporation into DNA by an average of 48 and 45%, respectively. Similar inhibitions were observed for porcine
and bovine kidney, porcine lung, and human KB cells. Thus, the effect of PSNE on the incorporation of [3H]thymidine into DNA of various cultured cells was either stimulatory to dermal cells or inhibitory to a variety of other
cell types, including skin epidermal cells. The stimulatory and inhibitory effects of PSNE were abolished by heating PSNE
for 5 min in boiling water before its addition to cell cultures. This suggests that macromolecular structure is important
in the action of PSNE.
This project was supported by a grant from the Research Advisory Board, University of Nevada, Reno, NV. 相似文献
17.
STABILITY OF DNA IN PURKINJE CELL NUCLEI OF THE MOUSE : An Autoradiographic Study 总被引:1,自引:0,他引:1
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Neurons of the mouse were labeled with [3H]thymidine during their prenatal period of proliferation. The 3H activity of the Purkinje cell nuclei was then studied autoradiographically 8, 25, 55, and 90 days after birth. The measured grain number per nucleus decreased by about 14% between the 8th and 25th postnatal days and then remained constant up to 90 days. There was no significant decrease of the 3H activity of the Purkinje cell nuclei after correction of the measured grain number per nucleus for increasing nuclear volume of the growing Purkinje cells and for the influence of [3H]β self-absorption in the material of the sections. Injection of a high dose of [3H]thymidine into young adult mice did not result in 3H labeling of either Purkinje or other neurons in other brain regions. The results agree with the concept of metabolic stability of nuclear DNA. "Metabolic" DNA could not be observed in these experiments. 相似文献
18.
19.
H. Gershman H. A. Crissman D. A. Carrino 《In vitro cellular & developmental biology. Plant》1981,17(2):143-149
Summary BALB/c 3T3 cells cultured as aggregates were examined by two independent techniques to determine whether or not cells accumulated
at a specific point in the cell cycle, and if so to determine the point at which they accumulate. Replating cells onto dishes
followed by pulse labeling with [3H]thymidine and autoradiography indicated that aggregate-cultured cells were in the same phase of the cell cycle as cells
cultured as confluent monolayers. Flow microfluorometry confirmed that 75% of the aggregate-maintained cells were arrested
in G0 or G1, with 25% distributed throughout the rest of the cell cycle. Labeling and mitotic indices of cells in aggregates were also
consistent with about 20 to 25% of the cells being in S+G2=M phases of the cell cycle at any time.
This work was supported by PHS Grant CA20323 and NSF Grant PCM 74-15092 to H. G., who is also a Harry H. Pinney Cancer Scholar. 相似文献
20.
Tritium-labelled uridine ([3H]UdR) perturbs progression of L1210 cells through the mitotic cycle. the main effect manifests as a slowdown or arrest of a portion of cells in G2 and is already observed 2 hr after addition of 0.5–5.0 μCi/ml of [3H]UdR into cultures. At 2.5–5.0 μCi/ml of [3H]UdR a slowdown of cell progression through S is also apparent. Additionally, there is an increase in the number of cells with DNA values higher than 4C in cultures growing in the presence of [3H]UdR for 8–24 hr. A pulse of [3H]UdR of 2 hr duration labels predominantly (95%) cellular RNA. the first cell-cycle effects (G2 slowdown) are observed when the amount of the incorporated [3H]UdR is such that, on average there are fewer than thirty-six [3H] decays per cell which corresponds to approximately 12–19 rads of radiation. the S-phase slowdown is seen at a dose of incorporated [3H]UdR twice as high as that inducing G2 effects. the specific localization of [3H]UdR in nucleoli, peripheral nucleoplasm and in cytoplasm, as well as differences in the kinetics of the incorporation in relation to phases of the cell cycle are discussed in the light of the differences between the effects of [3H]UdR and [3H]thymidine. Mathematical modelling of the cell-cycle effects of [3H]UdR is provided. 相似文献